Means for qualitative and quantitative analysis of microbial populations potentially present in a sample

ABSTRACT

This invention relates to means of qualitative and quantitative analysis of microbial populations potentially present in a sample. These means notably comprise the use of at least one RNA-targeted oligonucleotide probe for in situ hybridization in whole cells; followed by the extraction of those probes which have become hybridized by separation from their target and elution from the microbial cells; as well as the detection and measurement of said extracted probes.

FIELD OF INVENTION

This invention may be generally described as a means of qualitative andquantitative analysis of microbial populations potentially present in asample. More specifically, it relates to a means of qualitative andquantitative analysis using RNA targeted oligonucleotide probes.

BACKGROUND

The analysis of microbial populations potentially present is requiredfor many types of solid and fluid samples. Some notable examples arethose samples obtained from a natural or biological environment such asnatural water or hot springs; samples taken from humans or animals suchas blood, urine, vaginal and intestinal flora; and samples from urban,agricultural and industrial environments such as food products,industrial water, industrial effluents, municipal wastewater, industrialsludge, fermentation media, aerosols, filters or air from airconditioning systems.

Various laboratory techniques have been developed for the qualitativeand quantitative analysis of microbial populations potentially presentin a given sample.

One familiar technique involves a count of the microorganisms thatdevelop after the sample (or an extract thereof) is cultured on variousselective nutrient media under standard conditions. This technique issimple but entails significant risks of errors and artifacts (lowspecificity of morphological criteria, inability to detect viable butnon-culturable microorganisms, inability to detect slow-growingmicroorganisms, need to maintain viability of bacteria betweencollection and enumeration). Moreover, this technique generally requireslonger than 24 hours to yield results.

A second technique, which entails the measurement of the activity of oneor more enzymes, allows a rapid quantification of populations of livingmicroorganisms (culturable microorganisms and/or microorganisms in aviable but non-culturable form). This technique can be used, inparticular, to monitor a set of populations, but does not achieve veryhigh levels of specificity or sensitivity.

A third technique using immunological probes often requires a growthstep and thus requires longer than 24 hours to yield results. Moreover,it frequently lacks both sensitivity and specificity (misidentificationmay occur due to cross-reactions).

The most recent techniques are based on the use of specific DNA probes,which are generally labeled to permit detection after hybridization withtheir targets. Two main categories of oligonucleotide probes have beendeveloped: those that target DNA and those that target RNA (ribosomalRNA or messager RNA).

DNA-targeted probes, although potentially highly specific, have thedrawback of low sensitivity due to the few copies of the target DNAgenes in each microbial cell. Although the use of PCR (polymerase chainreaction) to amplify the target DNA sequences before detection cancompensate for the lack of sensitivity of the DNA probes, it has severaldrawbacks of its own: for example, the presence of inhibitors can leadto false-negative reactions, while carry-over or similar contaminationcan lead to false-positive reactions. In contrast, the use ofRNA-targeted probes prevents from such drawbacks. In particular, becauseof the large number of copies of rRNA that occur naturally in amicroorganism (actively growing cells may contain 10⁴ ribosomes, each apotential probe target), the use of rRNA-targeted probes does notrequire the amplification step, thereby overcoming the constraints andartifacts associated therewith. The advantage of targeting rRNA is thatabout 85-90 percent of the total RNA in a typical cell is rRNA.

The hybridization of RNA-targeted probes can be achieved either aftercell lysis, extraction and purification of the total nucleic acids ofthe sample, or in situ on whole cells, generally after fixation(permeabilization) of the membrane (or wall) of the microorganismspotentially present in the sample.

However, cell lysis and the ensuing extraction and purification of thenucleic acids particularly total RNA, are delicate and time-consumingmanipulations that require costly apparatus, trained personnel andstrict experimental conditions, notably the prevention of contaminationby nucleases during the procedure. This technique further implies theuse of a solid support, such as a nylon membrane, onto which thepurified nucleic acids are immobilized in such a way one candiscriminate between them (e.g. dot-blot, slot-blot). It most generallyalso implies the use of radioactive probe labels, the handling of whichrequires special care. The cell lysis technique for RNA hybrididizationis therefore ill-suited to use in routine analysis either in industry orin biological laboratories.

In situ hybridization in whole cells overcomes the need for preliminaryextraction of the target nucleic acids by cellular lysis with all itsassociated disadvantages. The FISH (Fluorescent In Situ Hybridization)process, which employs fluorescence-labeled rRNA probes, is one existingin situ technique. This type of technique, generally involvingfluorescence microscopy, provides a fast and sensitive qualitativeanalysis on many types of sample. Today, rRNA-targeted probes thushybridized in situ with their target within whole cells can bequantified directly on the sample (flow cytometry, microscopy), althoughthe method is not entirely satisfactory: quantification directly on thesample is technically costly, time-consuming, requires trained personneland does not permit an accurate quantification of hybridized probes whenthe sample is complex and non-uniform (e.g. floc or aggregates formed byfilamentous bacteria in sewage treatment sludge; samples containingnaturally fluorescent microorganisms). As a result, the technique of insitu hybridization in whole cells using fluorescence-labeledoligonucleotide probes has, to date, remained an essentially qualitativetechnique that does not provide reliable quantitative results.

To meet the need for industrial-caliber performance on samples that canbe complex and/or non-uniform, this invention provides a means foranalyzing, both qualitatively and quantitatively, the microbialpopulations potentially present in a biological sample, said meansovercoming the disadvantages of prior art techniques

SUMMARY OF THE INVENTION

The object of this invention is, therefore, a method of qualitative andquantitative analysis of the microbial population(s) potentially presentin a sample, characterized in that it comprises:

contacting the microorganisms potentially present in said sample with atleast one RNA-targeted oligonucleotide probe, hereafter called specificprobe, able to target a desired microbiological population, underconditions favourable to in situ hybridization in whole cells,

extracting, by separation from their target and elution outside saidcells, those probes which have become hybridized,

detecting the extracted probes and measuring the amount thereof or theirrespective amounts.

The present invention thus advantageously enables the extraction of saidprobes without destruction of said cells.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “microbiological population” (or“microbiological domain”) means the set of microorganisms that a givenprobe is able to recognize by recognition of an RNA target sequencepresent in each member of said set. The approach is based on RNA targetsequence present in each member of said set. The approach is based onoligonucleotide hybridization probes complementary to RNA sequences thatare diagnostic for selected phylogenetic groups which correspond, tovarying degrees, to a target region typical of a type of a microorganismor a whole group of microorganisms. Any probes enabling said contactingstep is appropriate for the implementation of the method according tothe invention. The choice of the specific probe(s) is directly relatedto the analysis desired for said sample. Probes can e.g. be composed ofoligonucleotide sequences that can distinguish between the primarykingdoms (eukaryotes, eubacteria, archaebacteria) and between closelyrelated organisms (the group of Ammonia-oxidizing β-Proteobacteria, thegenus Nitrobacter or Acinetobacter or the species Fibrobacterintestinalis, the species Escherichia Coli). Probes with finerphylogenetic resolution can be derived by using the existing collectionsof RNA sequences. Many examples of such RNA-targeted probes aredescribed in the prior art such as patents or patent applications,scientific publications e.g. Los Reyes et al. 1997, Appli. Environ.Microbiol. Vol. 63 No. 3 p.1107-1117; Mobarry et al. 1996, Appli.Environ. Microbiol. Vol. 62 No. 6 p.2156-2162; Wagner et al. 1994,Appli. Environ. Microbiol. Vol. 60 No. 3 p.792-800; Kane et al. Appli.Environ. Microbiol. Vol. 59 No. 3 p.682-686. Other examples of suchprobes can also be designed by the person skilled in the art.Advantageously probes are those which target ribosomal RNA (rRNA).Examples of such advantageous probes include Nb 1000 (SEQ ID No. 1) andNso 1225 (SEQ ID No. 2).

The method of the invention gives particularly accurate quantitativeresults when the cell numbers in said sample are equal to or greaterthan approximately 10³ or 10⁴ cells per ml. If desired, themicroorganism concentration of a liquid sample can be increased byfiltration or any other technique prior to implementing the method ofthe invention.

In a preferred arrangement of the invention, said microorganismspotentially present in the sample are also contacted with at least oneprobe, hereafter called “universal probe”, serving to normalize theresults obtained with probes targeting specific phylogenetic groups ofmicroorganisms (“specific probes”). The amount of a specific probe insaid sample may then be expressed as a ratio of the amount of saiduniversal probe. Such an universal probe may thus enable the expressionof e.g. the specific target rRNA as a percentage of the total rRNA.Examples of such “universal probes” include probes specific for anymicroorganism, or probes specific for bacteria, or for eukaryotes. Such“universal probes” are well-known in the art and any of them can be usedas long as it enables said contacting step, and allows the desired“specific probe” normalization. Such a “universal probe” is used in themethod according to the invention similarly as a “specific probe”, andaccordingly is advantageously a rRNA-targeted probe.

It may be advantageous to extract the microorganisms potentially presentin said sample therefrom, in particular by centrifugation, prior toproceeding with any step of the method of the invention. One reason toproceed in this manner is to remove the background noise that a sampleof complex composition can generate. Another reason may be to place intosolution the microorganisms potentially present in a solid, gazeous orviscous sample.

According to one embodiment of the invention, said contacting step isperformed after the cells are made to undergo a fixation step (orpermeabilization step) essential for maintaining their morphologicalintegrity, and which makes the microorganisms potentially present insaid sample permeable to short oligonucleotide probes (ca 15-25nucleotides). This fixation step allows the probes to penetrate insidethe microbial cells without affecting the integrity thereof, therebyattaining their target or targets in situ. Where applicable, said sampleis homogenized prior to said fixation step in order for said at leastone probe to have access to all microbial populations potentiallypresent in the sample.

Said fixation is advantageously achieved by incubating said cells in aparaformaldehyde solution that is less than 10%, preferably around 4%,for 3 to 12 hours at 4° C. This fixation procedure is more particularlyadapted to Gram-negative bacterias. For certain Gram-positive bacterias,said fixation step may be achieved by incubating said cells in a 100%ethanol solution.

Following fixation, said cells can be recovered by e.g. centrifugationand stored until use at −20° C. in a buffered solution at a pH of about7 (PBS buffer, for example) containing approximately 60% ethanol.

In a preferred arrangement of this embodiment of the invention, saidfixation is followed by a dehydration step (or drying step) prior tosaid contacting phase. Said dehydration step can thus be carried out byplacing said sample in contact with at least one ethanol solution,preferably with a series of ethanol solutions of increasingconcentration, for example by placing the sample in a 70%, 80% and then95% ethanol solution.

Advantageously, said contacting phase is performed by placing the samplein contact with said at least one probe in the presence of a solutionhereafter called “hybridization solution”, which comprises a denaturingagent such as sodium dodecyl sulfate (SDS) at a concentration in a0.001-0.1% range, preferably on the order of 0.01%; Tris-HCl, pH ofabout 8 at a concentration in a 0.001-0.1M range, preferably on theorder of 0.02M; and a salt such as sodium chloride at a concentration ina 0.1-1.5M range, preferably on the order of 0.9M. Such a contacting isadvantageously performed for an incubation time comprised between about10 minutes and about 2 hours, and at an hybridization temperature, whichis preferably the optimal temperature. For each oligonucleotide probe,the hybridization conditions (temperature; concentration of salts anddenaturing agents) can be indeed optimized so as to improve thespecificity of the oligonucleotide probe for the corresponding RNAsequences found in the target cells. When a plurality of oligonucleotideprobes is used simultaneously, these hybridization conditions can bechosen so as to take into account the optimal conditions peculiar toevery probe.

It is very advantageous for the extraction of said at least one probe tobe performed following the removal of excess and unbound probe or ofnon-specifically associated probe material placed in contact, notably bywashing with a solution hereafter called “wash solution”. Such a “washsolution” advantageously comprises a denaturing agent such as sodiumdodecyl sulfate (SDS) at a concentration in a 0.001-0.1% range,preferably on the order of 0.02%; tris-HCl pH of about 8 at aconcentration in a 0.001-0.1M range, preferably on the order of 0.02M,and a salt such as sodium chloride at a concentration in a 0.01-0.9Mrange, preferably on the order of 0.1M. The formulation of the <<washsolution>> (e.g. salt and denaturant nature and/or concentration) isadjusted so as to achieve the appropriate stringency; i.e. thestringency necessary to the removal of non-specifically associatedprobe. Thanks to such a washing step, the extraction step will beperformed only on those probes which have become effectively hybridizedto the desired target(s).

According to a preferred embodiment of the invention, said extraction isperformed by placing the microorganisms potentially present underconditions to denature enabling the denaturation of every probespecifically associated with its target sequence, notably in thepresence of a probe-target denaturing agent such as one that willseparate duplex DNA/DNA or DNA/RNA, and in particular the probe—targetduplex under consideration, and at a temperature higher than the meltingtemperature of the probe under consideration, notably at a temperatureof about 100° C. According to a particularly preferred embodiment of theinvention, said denaturing agent is formamide. Said extraction is thenperformed by incubating said microorganisms in formamide at 100° C. for10 minutes using a controlled temperature incubator. The supernatant maythen be recovered for quantification, e.g. by centrifugation. To improvedetection, said extracted probes can be concentrated notably using aSpeed-Vac® prior to measuring the corresponding amount of each probe.

The detection of a target-hybridized probe and the measurement of itsamount thus give a qualitative and quantitative analysis of the set oftarget-microorganisms present in the sample. It is advantageous toperform said detection and amount measurement of the extracted probes bydetection and amount measurement of a label associated with orincorporated into each of the contacted probes, such as a radioactive(³²P, ³⁵S, ¹²⁵I), chemiluminescent or fluorescent label. The respectiveamounts of probes are then measured by quantitation of the correspondinglabel. It is particularly advantageous to use a fluorescent label,notably fluorescein which can be easily quantified using a fluorescencespectrophotometer.

Different probes, e.g. specific probe(s) and/or universal probe(s), canbe placed in separate samples, or in the same sample. In the lattercase, it is possible to distinguish each probe used from the othersduring the detection step, for example by giving to each one its ownspecific label (e.g. different fluorochromes).

The method of the invention can be applied to a variety of samples.Samples for which an analysis using the method of the invention is ofparticular interest include those taken from fluids such as naturalwater, industrial water, industrial effluents, municipal wastewater,industrial sludge, thermal mud, food liquid or gel, fermentation medium,air, gas, aerosol; samples from a building ventilation duct, airconditioning duct; samples from edible solid, soil; samples from medicalapparatus; human or animal samples such as blood, urine, vaginal orintestinal flora.

The method of the invention utilizes neither microbiological culture,nor microscopy, nor an in vitro amplification step (like PCR) and doesnot require any cell lysis step. It is reproducible, simple, fast (lessthan 3 hours), low-cost and does not require specially trainedpersonnel. The method of the invention offers the additional advantageof being easy to automate. The method of the invention notably providesa qualitative and quantitative measurement of the microbiological orsanitary status of said sample and, consequently, of the product fromwhich said sample is taken. The method of the invention can thereforeadvantageously be combined with an alarm function relating to thequality, safety and/or sanitary monitoring of the product from which thesample is taken, notably as part of an industrial production line.

When the threshold value or set point is exceeded, the method of theinvention permits the corresponding quality, safety and/or sanitaryalarm to be triggered. It also permits the automatic or feedback controlof a microbiological removal or enrichment process.

This invention also relates to the application of said method to invitro diagnostics of infectious diseases.

Beyond applications of the “status or condition measurement” and “alarm”types, this invention relates in particular to the application of saidmethod for the automatic or feedback control of microbiologicalprocesses such as methane fermentation of liquid manure, treatment oforganic effluents, sewage treatment processes such as activated sludgetreatment; or to the automatic or feedback control of a process aimed atremoving or preventing the growth of microorganisms.

Thus, the method of the invention may be advantageously applied to thedetection of foam formation during the implementation of activatedsludge processes and/or to the feedback control of a process aimed atremoving or preventing the development of such foams.

Other features and advantages of the invention will further becomeapparent in the following exemplary embodiments, which are given forillustrative and non-limitating purposes.

EXAMPLE 1

Qualitative and Quantitative Analysis of a Sample of Sewage TreatmentReactor Effluent

a) Fixation Step

Samples of effluent from sewage treatment activated sludge reactors aremixed and then washed three times using a phosphate buffer solution (PBSphosphate-buffered saline) at pH 7. The sample is then incorporated intothree volumes of a 4% paraformaldehyde solution and incubated for 3 to12 hours at 4° C. Following centrifugation the supernatant is removedand the sample is again mixed with a phosphate-buffered saline solution(PBS) at pH 7. An equal volume of ethanol is added and the sample can bestored at −20° C. until use.

b) Dehydration Step

The fixed sample is centrifuged after adding 1 ml of 70% ethanol overthe residue and resuspending the cells. The mixture is centrifuged for 5minutes then the supernatant is removed. This procedure is repeated with80% ethanol and then again using 95% ethanol.

c) Hybridization Step

A water bath is prepared at the hybridization temperature required bythe probe being used (the temperature depends on the length and sequenceof the probe). In the example reported here, the following probes wereused:

Probe Nb 1000 specific to the Nitrobacter genus, with sequence SEQ IDNo. 1: 5′ TGCGACCGGTCATGG 3′

Probe Nso 1225, specific to Ammonia-oxidizing β proteobacteria, withsequence SEQ ID No. 2: 5′ CGCCATTGTATTACGTGTGA 3′

Probe S Univ-1390, a universal probe for any microorganism, withsequence SEQ ID No. 3: 5′ GACGGGCGGTGTGTACAA 3′, and

Probe S Bac338, specific for bacteria, with sequence SEQ ID No. 4: 5′GCTGCCTCCCGTAGGAGT 3′.

These probes were synthesized, purified by High Performance LiquidChromatography (HPLC), then fluorescein-labeled at the 5′ end. They areavailable from Operon Technologies of Alameda, Calif. (USA) or, inFrance, from the Genset company based in Paris (among others).

The cells obtained from the dehydration step are resuspended in 400 μLof a hybridization solution comprising (for 10 mL): NaCI 5M 1.8 mL;Tris-HCl 1M 200 μL; SDS (sodium dodecyl sulfate) 5 μL; distilledexcipient water 8 mL, for ten mL. After each probe is labeled by afluorochrome, the necessary quantity of each probe is added (here, 1.5nanomoles). The cells in the hybridization solution in contact with theprobes are incubated for 10 minutes to 2 hours at the hybridizationtemperature. The hybridization samples are centrifuged and supernatantsare removed.

d) Washing Step

Following hybridization, the cells are washed twice for 15 minutes eachtime at the hybridization temperature, in a buffered washing solutioncomprising, for 50 mL, NaCl 5M 1 mL; Tris-Hcl 1 M 9 mL; SDS 20% 50 μL.The formulation of the <<washing solution>> (e.g. salt and denaturant)is adjusted according to need of achieving appropriate stringency, i.e.removal of non-specifically associated probe.

e) Extraction of the Fluorescence by Elution

300 μL of formamide heated to 100° C. is added to the samples obtainedfrom the washing step, and the residue is gently resuspended. Each tubeis placed in 100° C. for 10 minutes, preferably using a controlledtemperature incubator. Centrifuge for 10 minutes. The supernatant isrecovered and stored in the dark until it can be analyzed byfluorescence spectroscopy. The fluorescence is quantified using aspectrofluorometer. The amounts measured in probes Nb1000 and Nso 1225correspond to the relative amounts of Nitrobacter bacteria andAmmonia-oxidizing β proteobacteria contained in the sample. Theseamounts are compared with those measured for universal probe S Univ-1390and bacteria probe S Bac 338.

This gives a percentage ratio (the relative proportion) of themicroorganisms contained in the sample, which are respectivelyNitrobacter and Ammonia-oxidizing β proteobacteria.

It is understood that this invention is not limited to the embodimentsdescribed and illustrated herein, but covers all variants thereof.

4 1 15 DNA Artificial Sequence Description of Artificial Sequenceprimer_bind 1 tgcgaccggt catgg 15 2 20 DNA Artificial SequenceDescription of Artificial Sequence primer_bind 2 cgccattgta ttacgtgtga20 3 18 DNA Artificial Sequence Description of Artificial Sequenceprimer_bind 3 gacgggcggt gtgtacaa 18 4 18 DNA Artificial SequenceDescription of Artificial Sequence primer_bind 4 gctgcctccc gtaggagt 18

What is claimed is:
 1. A method of qualitative and quantitative analysisof microbial population(s) comprising: providing a sample containingmicroorganisms, contacting the microorganisms present in the sample withat least one specific probe to form a sample with a probe-target complexin situ hybridization in whole cells, wherein the specific proberecognizes a RNA target sequence, contacting the sample with the probetarget complex with a wash solution to remove excess specific probes ornon-specific probes from the sample with the probe target complexthereby providing a washed sample, adding a denaturing agent to thewashed sample to extract the specific probes from the probe-targetcomplex, and detecting the extracted probes and measuring the amountthereof or their respective amounts to provide the qualitative andquantitative analysis of the microorganisms in the sample.
 2. A methodaccording to claim 1, wherein said at least one specific probe is chosenamong the group consisting of SEQ ID NO: 1 and SEQ ID NO:
 2. 3. A methodaccording to claim 1, further comprising contacting said microorganismspresent in said sample with an universal probe to normalize results. 4.A method according to claim 3, wherein said universal probe is chosenamong the group consisting of SEQ ID NO: 3 and SEQ ID NO:
 4. 5. A methodaccording to claim 3 wherein said specific or said universal probe is amRNA-targeted probe.
 6. A method according to claim 1, furthercomprising extracting said microorganisms in said sample bycentrifugation.
 7. A method according to claim 1, further comprisingfixing of said whole cells prior to contacting the microorganisms withthe at least one specific probe.
 8. A method according to claim 7,wherein fixation of the cells is achieved by incubation of the cells ina fixation solution of less than 10% paraformaldehyde for 3 to 12 hoursat 4° C.
 9. A method according to claim 7, wherein said fixation isfollowed by a dehydration step, prior to said contacting themicroorganisms with the at least one specific probe.
 10. A methodaccording to claim 9, wherein the dehydration is performed by placingsaid sample in contact with at least one ethanol solution.
 11. A methodaccording to claim 1, wherein said contacting the microorganisms withthe at least one specific probe includes placing said sample in contactwith said specific probe in the presence of a hybridization solutioncomprising a denaturing agent at a concentration of from 0.001% to 0.1%,Tris-HCl with a pH of about 8 at a concentration of from 0.001 M to 0.1M, and a salt at a concentration of from 0.1 M to 1.5 M.
 12. A methodaccording to claim 1, wherein contacting the microorganisms with the atleast one specific probe includes an incubation time of about 10 minutesto about 2 hours, and at an optimal hybridization temperature.
 13. Amethod according to claim 1, wherein the wash solution comprises a saltat concentrations appropriate for achieving the stringency necessary forthe removal of non-specifically associated probe.
 14. A method accordingto claim 1, wherein extracting of the hybridized probes includesextracting at a temperature higher than the melting temperature of thespecific probe under consideration.
 15. A method according to claim 14,wherein extracting of the hybridized probes includes adding formamide tothe washed sample.
 16. A method according to claim 1, wherein saidextracted probes are concentrated prior to the measurement of the amountthereof or of their respective amounts.
 17. A method according to claim1, wherein said detecting and measuring the amount of the extractedprobes includes detection and quantification of a label associated withor incorporated into the extracted probes, wherein the label is selectedfrom a radioactive label, a chemiluminescent label or a fluorescentlabel.
 18. A method according to claim 1, wherein said sample is takenfrom fluids selected from natural water, industrial water, industrialeffluent, municipal wastewater, industrial sludge, thermal mud, foodliquid or gel, fermentation media, air, gas, aerosol, a sample takenfrom a building ventilation duct or air conditioning duct, a sample offood solid, a sample of soil, a sample from medical apparatus, or is ahuman or animal sample selected from blood, urine, vaginal or intestinalflora.
 19. A method according to claim 1, wherein said method is used incombination with a process for triggering an alarm in connection withquality, safety and or sanitary monitoring of the product from whichsaid sample has been obtained.
 20. A method according to claim 1,wherein said method is used in in vitro diagnosis of an infectiousdisease.
 21. A method according to claim 1, wherein said method is usedin the automatic or feedback control of a microbiological process.
 22. Amethod according to claim 1, wherein said method is used in theautomatic or feedback control of a process relating to the removal orprevention of the development of microorganisms.
 23. A method accordingto claim 1 wherein said method is applied in the detection of foamformation during the implementation of activated sludge processes and/orthe feedback control of a method relating to the removal or preventionof the said foams.
 24. A method according to claim 10, wherein thedehydration comprises a series of ethanol solutions of increasingconcentrations.
 25. A method according to claim 11, wherein theconcentration of said denaturing agent is about 0.01%, the concentrationof said Tris-HCl is about 0.02 M, and the concentration of said saltabout 0.9 M.
 26. A method according to claim 11, wherein said denaturingagent is sodium dodecyl sulfate and said salt is sodium chloride.
 27. Amethod according to claim 13, wherein said denaturing agent is sodiumdodecyl sulfate and said salt is sodium chloride.
 28. A method accordingto claim 17, wherein said label is fluorescein.
 29. A method accordingto claim 8, wherein said fixation solution contains about 4%paraformaldehyde.
 30. A method according to claim 14, wherein saiddenaturing agent comprises formamide.